1. Field of the Invention
This invention is directed to the discovery that, within the polynucleotide sequence of papillomavirus (PV), there occur segments of nucleotide sequences which are useful in distinguishing and differentiating between PV types and species. These regions within the genome, the variable regions, are specific to the particular PV type and form the basis for production of DNA probes, antibodies (labelled and unlabelled), polypeptide segments, labelled polypeptide segments, and vaccines which are specific for a given particular PV.
In another aspect of the present invention, it has also been discovered that the polynucleotide sequence of all PVs contains a highly conserved region that is characteristic of all PVs and forms the basis for production of antibodies (labelled and unlabelled) and polypeptide segments (labelled and unlabelled), which are PV genus-specific, i.e. recognize and distinguish any PV and differentiate over non-PV viruses and viral products. This latter DNA sequence is termed "genus specific".
2. Description of the Background Art
The search for a virus involved in the induction of human cancer has been frustrating. A number of viruses have gained renewed interest as possible human cancer viruses; one such virus is the human papillomavirus (HPV). Although HPV was one of the first viruses to be visualized by the electron microscope, little information was available on the biology of the virus until recently. Early studies failed to provide direct indication that HPV may be associated with malignancy (these studies have been reviewed by Roson et al., Bacteriol. Rev., 31: 110-131 (1976)). However, as early as the 1930's, experimental evidence indicated that the cottontail rabbit papillomavirus (CRPV) was oncogenic in its host species (Rous et al., J. Exp. Med., 65: 523-548 (1935)). Further, other animal papillomaviruses have been shown to produce tumors in laboratory animals and some have been shown to be capable of morphologic transformation of cells in culture (Olson et al., Arch. Environ. Health, 19: 827-837 (1969)). However, neither experimental transmission of HPV to laboratory animals nor a tissue culture system permissive for virus replication or expression of biological activity has been successful. Accordingly, research on HPV has been limited to, for the most part, physical and chemical characterization of virions obtained from papillomas.
The inability to define a system permissive for replication of HPV virus in culture has been circumvented to some extent by molecular cloning of viral DNA sequences which permit detailed molecular analysis. Further, multiple, minimally related virus types with anatomic site preference have been discovered and HPV antigens and DNA in lesions with malignant potential have been detected. Previously, the causes of these lesions have been attributed to other infectious agents or some other unknown etiology. These developments have rendered the study of HPV attractive from the standpoint of viral oncogenesis.
It is known that the members of the papillomavirus genus are small (50-55 nm diameter), unenveloped viruses with an icosahedral symmetry. Their genome consists of a circular double-stranded DNA molecule containing about 8,000 base pairs (8 kb). The virions may be easily isolated from papillomas which are virus-positive by electron microscopy, by mechanically disrupting the epithelium followed by a series of differential centrifugation steps and banding in cesium chloride. In most preparations, two bands may be visualized, one at 1.33 gm/ml and the other at 1.29 gm/ml. It is believed that the former represents "full" virions which contain DNA, while the latter is composed of "empty" virus shells. The DNA may be isolated by rupture of virions with ionic detergent and removal of protein by organic extraction. The resultant DNA preparation generally contains two forms of DNA, a supercoiled fraction (Fo I) and a nicked-circular form (Fo II).
Five different viruses have been identified that infect cattle, i.e. the bovine papillomaviruses (BPV). These viruses have different degrees of DNA sequence homology, antigenic relatedness, tissue specificity (cutaneous or mucosal surfaces), and induce different types of lesions (fibropapillomas or papillomas).
The human papilloma viruses show even greater diversity. Eighteen different HPV types have now been described in the literature, and it is anticipated that additional types will be discovered. To be classified as a new virus type, there cannot be more than 50% DNA sequence homology under standard conditions of hybridization to previously typed viruses; a virus DNA which hybridizes to greater than 50% sequence homology to a given virus type is considered a subtype (Coggin et al., Cancer Res., 39: 545-546 (1979)). Standard hybridizations are run 25.degree. C. below the melting temperature of the DNAs (Tm-25.degree. C.) which allows for about 17% base mismatch (Laird et al., Nature, 224: 149-154 (1969)).
It now appears that the HPVs may be grouped with respect to sequence homology and site of infection as set out in the following Table 1.
TABLE 1 ______________________________________ The Human Papillomaviruses (HPV) % HOMOLOGY SITE LESION WITHIN GROUP ______________________________________ CUTANEOUS HPV-1 Plantar Wart &lt;1% HPV-2 Common Wart HPV-4 Plantar Wart HPV-7 Butchers' Common Wart HPV-3 Flat Wart &lt;35% HPV-10 Flat Wart HPV-5 EV* 5 to 39% HPV-8 EV HPV-12 EV HPV-14 EV HPV-9 EV 5 to 19% HPV-15 EV HPV-17 EV MUCOCUTANEOUS/MUCOSAL HPV-6 Genital Wart 3 to 25% HPV-11 Laryngeal Papilloma HPV-13 Focal Epithelial Hyperplasia HPV-16 Cervical Carcinoma &lt;1% HPV-18 Cervical Carcinoma ______________________________________ *Pityriasis-like lesions of epidermodysplasia verruciformis
Viruses infecting cutaneous surfaces are more likely to have some degree of homology to other HPVs infecting the skin than those infecting mucosal surfaces. Further, although a particular virus type is preferentially associated with a given lesion, it may, on occasion, be found in other lesions. As reported by Jensen et al., Lab. Invest., 47: 491-497 (1982), HPV-1 is associated with about 85% of plantar warts but HPV-2 has also been detected in a small percentage of plantar warts and vice versa. Of special interest is a group of human papillomaviruses, the types 3, 5, 8-10, 12-15, and 17. These viruses are found to be associated with individuals with epidermodysplasia verruciformis (EV), a rare recessive disorder characterized by generalized pityriasis-like lesions or flat warts (Jablonska et al., Cancer Res., 32: 583-589 (1972)). With the exception of types 3 and 10, this group of HPVs have not been detected in warts from healthy individuals, having only been identified in lesions from immunosuppressed renal allograft recipients (Lutzner et al., J. Invest. Dermatol., 75: 353-356 (1980)). It has also been reported that lesions containing HPV-5 and HPV-8 frequently undergo malignant conversion when present on sun-exposed areas. Hybridization analysis of primary and metastatic cancers have shown the presence of HPV-5 and HPV-8 DNA sequences within these tumors (Orth et al., Cell Prolif., 7: 259-282 (1980); Ostrow et al., Proc. Natl. Acad. Sci., 79: 1634-1638 (1982); and Pfister et al., Rev. Physiol. Biochem. Pharmacol., 99: 111-181 (1983)).
Recent studies using nonstringent hybridization conditions have demonstrated that DNA sequences are conserved among the genomes of each HPV genome as well as between HPVs and other animal papillomaviruses (Heilman, C. A., J. Virol., 36: 395-407 (1980); and Law, M. F., J. Virol., 32: 199-207 (1979)). Thus, it is now possible to use a PV-specific DNA probe to examine the DNA prepared from a putative PV-induced lesion to search for related sequences by using nonstringent hybridization conditions. This approach has been demonstrated to be successful for HPV DNA sequences of unknown HPV types as well as in the DNA prepared from anogenital warts (Krzyzek, R. A. et al., J. Virol , 36: 236-244 (1980)) and from juvenile laryngeal papillomas (Lancaster, W. D., Intervirology, 15: 204-212 (1981)).
In spite of the absence of a suitable tissue culture system for analysis of PV genomes, considerable insight into the possible functions of the virus genomes has been developed as a result of DNA sequence analysis, substantial quantities of the DNA being produced by molecular cloning techniques. The nucleotide sequences for bovine papillomavirus type 1 (BPV-1) (Chen et al., Nature, 299: 529-534 (1982)), HPV-1a (Danos et al., Embo. J., 1: 231-236 (1982)), and HPV-6b (Schwartz et al., Embo. J., 2: 2341-2348 (1983)) are now known. Known as well are the DNA sequences for HPV-11, HPV-16, and deer papillomavirus (DPV).
It has also been demonstrated that the papilloma viruses are related antigenically. Although antisera raised against intact viral particles of a specific HPV are type specific and will not react in tissues infected by another HPV type, antisera raised against PV virions that have been disrupted by heat and detergent will cross-react with capsid antigens of all HPVs, as well as PVs of other animal species (Jensen, A. B. et al., JNCI, 64: 495-500 (1980)). This information has led to the speculation that there are conserved amino acid sequences in the major capsid proteins of papillomas viruses that are responsible for this genus-specific antigenic cross-reactivity, and that these conserved amino acid sequences are not exposed on the surface of the virion particles (Howley, P. M., Arch. Pathol. Lab. Med., 106: 429-432 (1982)).
The use of antisera raised against the common PV antigens, then, is useful for identification of cells that are productively infected by any HPV, known or unknown. This cross-reacting antisera has allowed pathologists and clinical investigators to identify HPVs as the etiologic agent of juvenile laryngeal papillomatosis (Costa, J. et al., Am. J. Clin. Pathol , 75: 194-197 (1981); and Lack, E. et al., Intervirology, 14: 148-156 (1981)) and of cervical flat warts (Kurman, R. J. et al., Am. J. Obstet. Gynecol., 140: 931-935 (1981); and Morin, C. et al., JNCI, 66: 831-835 (1981)). However, the antisera are not useful or suitable for identifying a specific HPV. Additionally, it is essential that the viral infectivity has progressed to the point that protein expression has occurred, the antisera not being useful for picking up early stages of infectivity or infectivity where protein expression has not occurred.
To date, little is known about the functional aspects of HPV genomes. More information is available regarding animal papillomavirus systems. Because the genomic organization is highly conserved among the papillomaviruses, it is reasonable to assume similar relationships with respect to genome structure and function between animal and human papillomaviruses. Since some of the animal papillomaviruses are highly oncogenic, it is likely that all or a portion of this function may be a property of some of the HPVs as well. Many HPV-induced lesions such as common warts, plantar warts, and flat warts are entirely benign and not clinically associated with progression to carcinomas. However, several HPVs are occasionally associated with subsequent development into squamous cell carcinomas, and HPV-5 has been associated with cutaneous carcinomas in patients with epidermodysplasia verruciformis. Juvenile laryngeal papillomatosis is caused by papillomavirus HPV-11. Rare cases of spontaneous progression to invasive squamosal carcinoma of the larynx in the absence of irradiation have been described by Runckel, D. et al., Am. J. Surg. Pathol., 4: 293-296 (1980). More frequently, following radiation therapy, progression of juvenile laryngeal papillomatosis progresses to squamous cell carcinoma. Anogenital warts (condoloma acuminata) is caused by a number of different HPVs. The literature contains reports of progression of some of these lesions to locally invasive squamous cell carcinomas (Zur Hausen, H., Curr. Top. Microbiol. Immunol., 78: 1-30 (1977)). Further, HPV-specific antigens have been detected in 50% of the cervical lesions interpreted as dysplastic. The clear epidemiologic association of cervical dysplasia to carcinoma in situ and invasive cervical carcinoma strongly suggests the role of at least HPV in this progression.
The existence of a large number of PVs, with more than one PV occurring in certain PV-induced lesions, the close association of papilloma and various carcinomas, as well as the non-cancer related medical problems resulting from virus-induced papillomas has produced an on-going ever-increasing need for a methodology to identify, with a high degree of certainty, the specific etiology of the papilloma. Thus, a need has continued to exist for a means for differentiating between the large number of PV types known to exist, as well as a means for further identifying as yet unknown PV types.
At the same time, it would be of substantial value, and a need has continued to exist, for the determination of the highly conserved nucleotide sequence which is common to all PVs and the "common antigen" which this highly conserved nucleotide sequence codes for. The determination of this sequence would be of substantial value in the production of antibodies which are cross-reactive with all PVs.
It is now known that productive papillomavirus infection of epithelial cells results in hyperplasia of cells in the spinous layer (acanthosis). Cells show an increase in size and in the number of desmosomes and tonofibrils. Other epithelial cells show degenerative changes with loss of tonofibrils, detachment of desmosomes, nuclear wrinkling, and cytoplasmic vacuolization. In the upper layers of the epithelium, these changes are more pronounced. Cells in the granular layer show nuclear degeneration, margination, and condensation of chromatin. Virions are evident in nuclei of degenerated cells in the keratin layer by electron microscopy and the frequently intercrystalline array.
Host immune responses to papillomavirus infection are not well understood but infection usually occurs in the young, followed by persistence of the wart for a variable period of time. After regression, the host is left immune to reinfection by the same virus. In humans, antibody response to HPV infection is characterized by the appearance of IgM prior to the onset of regression. Just subsequent to regression, both IgM and IgG antibodies are present; long after regression, only IgG is detectable (von Krough, G. J., Dermatol., 18: 195-204 (1979)). This conversion from IgM to IgG has been observed in cattle experimentally infected with BPV as well (Lee, K. P. et al., Cancer Res., 29: 1393-1397 (1969)).
It would appear that rejection of papillomas is closely associated with cell-mediated immunity. In cattle experimentally infected with papillomavirus, regression is preceded by infiltration of mononuclear leukocytes, mainly lymphocytes. This occurs generally in perivascular areas, but also as a diffuse scattering throughout the papilloma (Lee, K. P. et al., J. Invest. Dermatol., 52: 454-464 (1969)).
In humans, regressing flat warts demonstrate a similar histological appearance with perivascular infiltration of mononuclear leukocytes in the upper dermis, with epidermal invasion sharply confined to the papilloma (Tagami, H. et al., J. Dermatol., 90: 147-154 (1974)). This simultaneous regression of multiple warts at distant sites suggest that cell-mediated immunity plays a major role in papilloma rejection. Regression of flat warts, however, has no effect on plantar or palmar warts in the same individual, indicating that regression is HPV type-specific (Berman, A. et al., Br. J. Dermatol., 99: 179-18 (1978)). A similar differential regression of warts has also been observed in cattle infected with multiple BPV types (Barthold, S. W. et al., J. Am. Vet. Med. Assoc., 165: 276-280 (1974)).